The Utility of Ultra-Deep RNA sequencing in Mendelian Disorder Diagnostics.

Clinical RNA-seq has become an essential tool for resolving variants of uncertain significance (VUS), particularly those affecting gene expression and splicing. However, most reference data and diagnostic protocols employ relatively modest sequencing depths (∼50-150 million reads), which may fail to capture low-abundance transcripts and rare splicing events critical for accurate diagnoses. We evaluated the diagnostic and translational utility of ultra-high-depth (up to ∼one billion unique reads) RNA-seq in four clinically accessible tissues (blood, fibroblast, LCL, and iPSC) using Ultima sequencing platform. After validating the performance of Ultima RNA-seq, we investigated how increasing depth affects gene and isoform detection, splicing variant discovery, and clinical interpretation of VUS. Deep RNA-seq substantially improved sensitivity for detecting lowly-expressed genes and isoforms. At ∼1 billion reads, near-saturation was achieved for gene-level detection, although isoform-level coverage continued to benefit from even deeper sequencing. In two clinical cases with VUS, pathogenic splicing abnormalities were undetected at ∼50 million reads but emerged at 200 million reads, becoming even more pronounced at ∼one billion reads. Using deep RNA-seq data, we constructed a novel resource, MRSD-deep, to estimate the minimum required sequencing depth to achieve desired coverage thresholds. MRSD-deep provided gene- and junction-level guidelines, aiding labs in selecting suitable coverage targets for specific applications. Leveraging deep RNA-seq data on fibroblast, we also built an expanded splicing-variation reference that successfully identified rare splicing events missed by standard-depth data. Our findings underscore the diagnostic and research benefits of deep RNA-seq for Mendelian disease investigations. By capturing rare transcripts and splicing events, ultra-high-depth RNA-seq can facilitate more definitive variant interpretations and enrich splicing-reference databases. We anticipate that cost-effective deep sequencing technologies and robust reference cohorts will further advance RNA-based diagnostics in precision medicine.